The field of human medicine has been dependent on the ability to detect changes caused by or in response to disease. Such changes provide means of diagnosis and offer insights to the targets for therapeutic compounds such as vaccines and medicines. A wide range of biological molecules can be used in medicine including nucleic acids, proteins, steroids, sugars and lipids. In this context, the ability to quantitatively detect such biomolecules using mass spectrometers has provided considerable advances in their study and application to human and veterinary disease, in environmental analysis and monitoring, and in food and beverage manufacturing. In particular the use of stable isotopes to provide synthetic quantitative references has been developed in isotope dilution mass spectrometry for monitoring of all classes of biomolecules. However, these methods have traditionally required an available synthetic standard which is not always possible.
Recently a range of chemical mass tags bearing heavy isotope substitutions have been developed to further improve the quantitative analysis of biomolecules by mass spectrometry. Depending on the tag design, members of tag sets are either isochemic having the same chemical structure but different absolute masses, or isobaric having both identical structure and absolute mass. Isochemic tags are typically used for quantitation in MS mode whilst isobaric tags must be fragmented in MS/MS mode to release reporter fragments with a unique mass. To date the isotopically doped mass tags have primarily been employed for the analysis of proteins and nucleic acids.
An early example of isochemic mass tags were the Isotope-Coded Affinity Tags (ICAT) (Gygi et al., Nature Biotechnology 17: 994-999, “Quantitative analysis of complex protein mixtures using isotope-coded affinity tags” 1999). The ICAT reagents are a pair of mass tags bearing a differential incorporation of heavy isotopes in one (heavy) tag with no substitutions in the other (light) tag. Two samples are labelled with either the heavy or light tag and then mixed prior to analysis by LC-MS. A peptide present in both samples will give a pair of precursor ions with masses differing in proportion to the number of heavy isotope atomic substitutions. Further examples of isochemic tags include the ICPL reagents that provide up to four different reagents.
Whilst isochemic tags allow a degree of improvement in the reproducibility of proteomic studies, this is achieved at the cost of increasing the complexity of the mass spectrum. To overcome this limitation, and to take advantage of greater specificity of tandem mass spectrometry the isobaric mass tags were developed. Since their introduction in 2000 isobaric mass tags have provided improved means of proteomic expression profiling by universal labelling of amine functions in proteins and peptides prior to mixing and simultaneous analysis of multiple samples. Because the tags are isobaric, having the same mass, they do not increase the complexity of the mass spectrum since all precursors of the same peptide will appear at exactly the same point in the chromatographic separation and have the same aggregate mass. Only when the molecules are fragmented prior to tandem mass spectrometry are unique mass reporters released, thereby allowing the relative or absolute amount of the peptide present in each of the original samples to be calculated.
U.S. Pat. No. 7,294,456 sets out the underlying principles of isobaric mass tags and provides specific examples of suitable tags wherein different specific atoms within the molecules are substituted with heavy isotope forms including 13C and 15N respectively. U.S. Pat. No. 7,294,456 further describes the use of offset masses to make multiple isobaric sets to increase the overall plexing rates available without unduly increasing the size of the individual tags. WO 2004/070352 describes additional sets of isobaric mass tags. WO 2007/012849 describes further sets of isobaric mass tags including 3-[2-(2,6-Dimethyl-piperidin-1-yl)-acetylamino]-propanoic acid-(2,5-dioxo-pyrrolidine-1-yl)-ester (DMPip-βAla-OSu).
Despite the significant benefits of previously disclosed isobaric mass tags there remains a need for further improvement both in the range of molecules that can be labelled with such tags, and also in the levels of multiplex analysis achievable. Accordingly, it is an aim of the present invention to provide a range of novel isobaric mass tags that specifically address the limitations of previously disclosed molecules.